a) Field of the Invention
The invention is directed to a method in microscopy, particularly fluorescence microscopy, laser scanning microscopy, fluorescence correlation microscopy and scanning nearfield microscopy, for examining primarily biological specimens, preparations and associated components. This includes methods for screening active ingredients (high throughput screening) based on fluorescence detection. Simultaneous examinations of specimens with multiple fluorophores in real time by means of simultaneous illumination of the specimen at a plurality of points on the specimen are therefore possible with overlapping fluorescence spectra even in three-dimensional structures of thick specimens.
b) Description of the Prior Art
A typical area of application of light microscopy for the examination of biological preparations is fluorescence microscopy (Pawley, “Handbook of Biological Confocal Microscopy”; Plenum Press 1995). For this purpose, determined dyes are used for specific labeling of cell parts.
The irradiated photons having a determined energy excite the dye molecules, through the absorption of a photon, from the ground state to an excited state. This excitation is usually referred to as single-photon absorption (FIG. 1a). The dye molecules excited in this way can return to the ground state in various ways. In fluorescence microscopy, the most important transition is by emission of a fluorescence photon. Because of the Stokes shift, there is generally a red shift in the wavelength of the emitted photon in comparison to the excitation radiation; that is, it has a greater wavelength. Stokes shift makes it possible to separate the fluorescent radiation from the excitation radiation.
The fluorescent light is split off from the excitation radiation by suitable dichroic beam splitters in combination with blocking filters and is observed separately. This makes it possible to show individual cell parts that are dyed with different dyes. In principle, however, multiple parts of a preparation can also be dyed simultaneously with different dyes which bind in a specific manner (multiple fluorescence). Special dichroic beam splitters are used again to distinguish the fluorescence signals emitted by the individual dyes.
In addition to excitation of dye molecules with a high-energy photon (single-photon absorption), excitation with a plurality of lower-energy photons is also possible (FIG. 1b). The sum of energies of the single photons corresponds approximately to a multiple of the high-energy photon. This type of excitation of dyes is known as multiphoton absorption (Corle, Kino, “Confocal Scanning, Optical Microscopy and Related Imaging Systems”; Academic Press 1996). However, the dye emission is not influenced by this type of excitation, i.e., the emission spectrum undergoes a negative Stokes shift in multiphoton absorption; that is, it has a smaller wavelength compared to the excitation radiation. The separation of the excitation radiation from the emission radiation is carried out in the same way as in single-photon excitation.
The prior art will be explained more fully in the following by way of example with reference to a confocal laser scanning microscope (LSM) (FIG. 2).
An LSM is essentially composed of four modules: light source, scan module, detection unit, and microscope. These modules are described more fully in the following. In addition, reference is had to DE19702753A1.
Lasers with different wavelengths are used in an LSM for specific excitation of different dyes in a preparation. The choice of excitation wavelength is governed by the absorption characteristics of the dyes to be examined. The excitation radiation is generated in the light source module. Various lasers (argon, argon/krypton, Ti:Sapphire lasers) are used for this purpose. Further, the selection of wavelengths and the adjustment of the intensity of the required excitation wavelength is carried out in the light source module, e.g., using an acousto-optic crystal. The laser radiation subsequently reaches the scan module via a fiber or a suitable mirror arrangement.
The laser radiation generated in the light source is focused in the preparation in a diffraction-limited manner by the objective through the scanner, scan optics and tube lens. The focus is moved in two dimensions in x-y direction over the specimen. The pixel dwell times when scanning over the specimen are mostly in the range of less than one microsecond to several seconds.
In confocal detection (descanned detection) of fluorescent light, the light emitted from the focal plane (specimen) and from the planes located above and below the latter reaches a dichroic beam splitter (MDB) via the scanner. This dichroic beam splitter separates the fluorescent light from the excitation light. The fluorescent light is subsequently focused on a diaphragm (confocal diaphragm/pinhole) located precisely in a plane conjugate to the focal plane. In this way, fluorescent light components outside of the focus are suppressed. The optical resolution of the microscope can be adjusted by varying the size of the diaphragm. Another dichroic blocking filter (EF) which again suppresses the excitation radiation is located behind the diaphragm. After passing the blocking filter, the fluorescent light is measured by a point detector (PMT).
When using multiphoton absorption, the excitation of the dye fluorescence is carried out in a small volume in which the excitation intensity is particularly high. This area is only negligibly larger than the detected area when using a confocal arrangement. Accordingly, a confocal diaphragm can be dispensed with and detection can be carried out directly following the objective (nondescanned detection).
In another arrangement for detecting a dye fluorescence excited by multiphoton absorption, descanned detection is carried out again, but this time the pupil of the objective is imaged in the detection unit (nonconfocal descanned detection).
From a three-dimensionally illuminated image, only the plane (optical section) coinciding with the focal plane of the objective is reproduced by both detection arrangements in connection with corresponding single-photon absorption or multiphoton absorption. By recording a plurality of optical sections in the x-y plane at different depths z of the specimen, a three-dimensional image of the specimen can be generated subsequently in computer-assisted manner.
Accordingly, the LSM is suitable for the examination of thick preparations. The excitation wavelengths are determined by the utilized dye with its specific absorption characteristics. Dichroic filters adapted to the emission characteristics of the dye ensure that only the fluorescent light emitted by the respective dye will be measured by the point detector.
Currently, in biomedical applications, a number of different cell regions are labeled simultaneously by different dyes (multifluorescence). In the prior art, the individual dyes can be detected separately based on different absorption characteristics or emission characteristics (spectra). For separate detection, an additional splitting of the fluorescent light of a plurality of dyes is carried out with the secondary beam splitters (DBS) and a separate detection of the individual dye emissions is carried out in various point detectors (PMT x).
Flow cytometers are used for examining and classifying cells and other particles. For this purpose, the cells are dissolved in a liquid and are pumped through a capillary. In order to examine the cells, a laser beam is focused in the capillary from the side. The cells are dyed with different dyes or fluorescing biomolecules. The excited fluorescent light and the backscattered excitation light are measured. The separation of the fluorescence signal of the specimen from the excitation light is carried out by means of dichroic beam splitters (MDB, see FIG. 2). The art is described in “Flow Cytometry and Sorting”, second edition, M. R. Melamed, T. Lindmo, M. L. Mendelsohn, eds., Wiley & Sons, Inc., New York, 1990, 81-107.
The size of the cells can be determined from the backscattered signal. Different cells can be separated and/or sorted or counted separately by means of the spectral characteristics of the fluorescence of individual cells. The sorting of the cells is carried out with an electrostatic field in different capillaries. The results, that is, for example, the quantity of cells with dye A in comparison to cells with dye B, are often displayed in histograms. The through-flow rate is typically about 10-100 cm/s. Therefore, a highly sensitive detection is necessary. According to the prior art, a confocal detection is carried out in order to limit the detection volume.
According to the prior art, line scanners, as they are called, are also used instead of point scanners (Corle, Kino, “Confocal Scanning Optical Microscopy and Related Imaging Systems”, Academic Press 1996). The basic construction essentially corresponds to that of an LSM according to FIG. 2. However, instead of a point focus, a line is imaged in the specimen and the specimen to be examined is scanned in only one direction (x or y). The image acquisition rate can be substantially increased by scanning a line instead of a point. Therefore, this scanning method can be used for observing high-speed processes in real time (real time microscopy). However, the optical axial resolution is reduced by a factor of approximately 1.4 compared with a point scanner.
In another arrangement for real time microscopy according to the prior art, the entire field to be examined is illuminated by an expanded light source. However, only special point patterns of the total field to be scanned are uncovered by a rapidly rotating disk. These methods are mostly known in technical literature as Nipkow disk methods (Corle, Kino, “Confocal Scanning, Optical Microscopy and Related Imaging Systems”, Academic Press 1996).
In another method according to the prior art, known as structured illumination (see FIG. 3), the modulation depth of the optical imaging of an amplitude structure (e.g., grating) is used as a criterion for the depth of field. The image of the periodic structure is distinguished by the frequency of the modulation and the phase position (image phase) of the modulation. Various projection scenarios can be obtained by means of a phase shift of the structure at right angles to the optical axis. Generally, at least three phase images PB are required at 0°, 120° and 240° in order to calculate depth-discriminated optical sections without stripes. These phase images (PB) are subsequently calculated to form a (confocal) optical section image in an image processor by the following formula:
                    I        Section            ⁡              (        x        )              =          Const      ·                                                                                                        (                                                                  I                        ⁢                                                  (                                                      x                            ,                                                                                                                  ⁢                                                          0                              ⁢                                                                                                                          ⁢                              °                                                                                )                                                                    ⁢                                                                                          -                                                                                          ⁢                                              I                        ⁢                                                  (                                                      x                            ,                                                                                                                  ⁢                                                          120                              ⁢                                                                                                                          ⁢                              °                                                                                )                                                                                      )                                    2                                +                                                      (                                                                  I                        ⁢                                                  (                                                      x                            ,                                                                                                                  ⁢                                                          120                              ⁢                                                                                                                          ⁢                              °                                                                                )                                                                    ⁢                                                                                          -                                              I                        ⁢                                                  (                                                      x                            ,                                                                                                                  ⁢                                                          240                              ⁢                                                                                                                          ⁢                              °                                                                                )                                                                                      )                                    2                                ⁢                                                                  +                                                                                                          (                                                            I                      ⁢                                                                                          ⁢                                              (                                                  x                          ,                                                                                                          ⁢                                                      0                            ⁢                                                                                                                  ⁢                            °                                                                          )                                                              ⁢                                                                                  -                                                                                  ⁢                                          I                      ⁢                                                                                          ⁢                                              (                                                  x                          ,                                                                                                          ⁢                                                      240                            ⁢                                                                                                                  ⁢                            °                                                                          )                                                                              )                                2                                                          ,where I(x, angle) describes the intensity at the respective pixel in the corresponding phase image.
It is simplest to carry out the recording of three or more phase images sequentially. In this connection, it is assumed that the specimen is not moved during the measurement of the images. The section images or section stacks which are calculated from the phase images in this way can be displayed subsequently on a standard PC and monitor by means of 3-D evaluating software. The spatial resolution along the optical axis depends on the wavelength of the light, the numerical aperture of the objective and the modulation frequency.
For a detailed description of the calculation algorithm, reference is had to T. Wilson, et al., “Method of obtaining sectioning by using structured light in a conventional microscope”, Optics Letters 22 (24), 1997.
Arrangements for screening dyes, for example, in so-called chip readers are similar in optical construction to laser scanning microscopes. However, they scan an appreciably larger image field for the investigation of macroscopic specimens, for example, screening of active ingredients on a biochip. The edge length of the scan fields amounts to about 10 mm. These scan fields can be achieved, e.g., by increasing the scan angle of the galvoscanner, by arranging the specimen in an intermediate image of the microscope arrangement or by a special objective arrangement (macroobjective) which images the intermediate image on the specimen in magnified manner.
According to the prior art, the separation of the excitation light from the light emitted by the specimen is carried out by spectral separation using Stokes shift by restricting the numerical aperture of the optics used for specimen illumination and detection or by splitting into different polarization directions.
Special dichroic beam splitters are used for the spectral separation of the excitation light from the light emitted by the specimen. As is shown in FIG. 3a, these dichroic beam splitters are usually constructed in such a way that they reflect the excitation light as efficiently as possible and transmit the light emitted by the specimen as efficiently as possible. The reflection factor (reflectivity) is shown as a function of wavelength. When using polarized excitation light, the minimum spectral bandwidth (s) of the reflected wavelength range is about 10 nm; the edge steepness (f) is usually greater than 5 nm. Therefore, according to the prior art, the light emitted by the specimen can be efficiently separated with a dichroic beam splitter when using an excitation wavelength. However, efficiency decreases when a plurality of dyes with a plurality of wavelengths are excited simultaneously (multifluorescence microscopy), since a spectral overlapping of the excitation light and the emitted light usually occurs. Further, a special beam splitter must be created each time when using different dyes with different absorption characteristics. In a widefield microscope, there is usually a broadband excitation of the specimen with light from a white light source with partial spectral overlapping of the excitation radiation and emitted radiation. Accordingly, the use of dichroic beam splitters according to the prior art results in a poor efficiency of separation of excitation light and emitted light.
The separation of excitation light from emitted light by restricting the numerical aperture of the specimen illumination optics (4 in FIG. 3b) can be carried out, for example, by illuminating the specimen with a restricted aperture, so that only the near-axis beams (1) travel in the direction of the specimen (2). Since the emission is carried out in all spatial directions, this light from the specimen (2) can be collected in the rest of the aperture area. The separation of the excitation light from the emitted light is carried out subsequently by a partially fully reflecting (black area) plane plate (3). The detection of the light emitted by the specimen is carried out in the radiating direction (5). The methods for dividing the numerical aperture known from the prior art (e.g., Pawley, Handbook of Confocal Microscopy, 1995) are disadvantageous in that the efficiency of detection and the optical resolution of the arrangement are worsened by the restriction of the aperture. These two parameters are connected in this regard. For example, a highly efficient separation is achieved at the expense of worsened optical resolution.
Problems of the Prior Art
In all of the methods according to the prior art, it is disadvantageous that the separation of the excitation light from the light emitted by the specimen is carried out in a wavelength-dependent manner, i.e., can not be adjusted in a flexible manner, or with a limited efficiency of typically 70% to 90%, depending on the required spectral characteristics and the quantity of illumination lines.